Vol. 57, No. 6
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1838-1841
0099-2240/91/061838-04$02.00/0 Copyright C) 1991, American Society for Microbiology
Molecular Structure of the Replication Origin of a Bacillus subtilis (natto) Plasmid, pUHI TOSHIO HARA,* SINICHIRO NAGATOMO, SEIYA OGATA, AND SEINOSUKE UEDAt
Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan Received 8 November 1990/Accepted 7 March 1991
The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for y-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promotor region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with repl4 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.
fermented soybean food, natto. Recently, electron microscope studies of the hybridization products of pLS11, which is detected in B. subtilis IFO 3022 and is 8.6 kb in size, with pUH1 revealed a heteroduplex molecule containing 1.11and 2.14-kb double-stranded DNA termini (6). We thought that these double-strand regions might play an important role in polyglutamate production and DNA replication. This communication reports that the 2.0-kb BstEII DNA fragment corresponds to the replication origin of Natto plasmid pUH1 and contains a 999-bp open reading frame, a promoter for the open reading frame, and a possible replication origin upstream of the promoter. Significant homologies were observed between the amino acid sequence predicted from the 999-bp open reading frame and those of the similar putative proteins encoded by the B. amyloliquefaciens plasmid pFTB14 (13) and Staphylococcus aureus plasmid pC194 (3, 7). To facilitate identification of the replication region of
In a previous paper (5), we reported that a 5.7-kb plasmid designated pUH1, which encodes the y-glutamyltranspeptidase gene responsible for y-polyglutamate production, was detected in a Bacillus subtilis (natto) strain isolated from a
I 1000
Fragment 500
1
Phenotype 1750
1
2 3 4 5 6 7 8 8 9 10 11
FIG. 1. Derivation of plasmids used in the present study. The chain of solid circles in the diagram indicates the DNA segment containing the Tmpr gene of B. subtilis TTK24. Heavy and thin lines represent the regions of pBR322 and pTL12, respectively. Double
2000 bp +
+1
r
~~~~~v v
B B
4.
FIG. 2. Structure and replication activity of the modified fragments of the 2.0-kb ori fragment. The open arrow in the restriction map indicates an open reading frame found in the sequence data (see Fig. 3). + and - indicate, respectively, ability and inability to replicate in the B. subtilis host. Modifications were made by deletion with restriction enzymes or insertion of a few base pairs (closed triangle), using the Klenow fragment, after digestion with the indicated restriction enzyme or by Bal31 digestion (B) from the BglII
lines represent the DNA fragment of pUH1. A, AatI; Bg, BglII; Bs, BstELL; E, EcoRI; H, HindIII; P, PstI.
* Corresponding author. t Present address: Department of Applied Microbiology and Technology, Kumamoto Institute of Technology, Ikeda, Kumamoto 860, Japan.
site of pBB2. 1838
VOL. 57, 1991
NOTES
1839
BstE II
5' -ggtaaccGGACCGTAGGGA 50
1TA GGATTAAGGAAGTTGACTCGCTCAGCGCCACCCGAACCCTTTCCGCACTCt.AACAAACCCGTTTGTTTGACGCCAACCG 150
°°
GGCAGGGAGCCCCCCGAAGAAGCGGGGGTTGGGGGGATTGAATGCTGGCATCCAACGGCCGTCCGTTGGTGGGTTTGGG 250
200
CAAAGCCAAGAACTGTTGCAAGGCTCGTTGAGAATAAAGAATGCTTTTCAGGATGCTTAGAATCGTTTCTGAGAGCTTC 300
AAATAAAAAAGATGACCTTTTATAGGGGGAAGCTCTTAAAATTGAATGTAGGGGCATTTAAACACGTTTAAAAATAAAA 350
400
AAAGCAGACTCTTTAGAGTCCGCCTTGTTATTTTTAACCCAGTGCTCCATTTTCGGCTGTTTGGAAATCTTTTGAGATG CCGAACCATCCATTTTCTTTTGTTCCATGAAAAAAGTGCTTTTGGATGCTTAAAAAGGCTTTTTCGTATAAAAAAAGCC 500
550
GATTTTTGAAAAAAAAATCTCCCTGCGGGGGAAGAATGGTTTTGATCTTTGGGTTTTAGGTTTTAAAAAAACCGGGCTG 650
600
TTTTCAGCCGGCTTTTTTTCGATTTTGGCGGAGCCGAAATCGGGTCTTTTCTTATCTTGATACTATATAGAAACATCTC 2
12
36
AAGGCGAAAAAATAGCCCCCATCCCTTATTTGTCAAGGGTTTGACGGCTTTGTTAGAAACTCCTTCCGCffl (SD) so '800 M3AAAGTGCCCACTAAAATAATAGAATGCTAGATTACTAG CTCA q~ TTTTTTGT6TCATGTATTCATCTGAAA 850 ATG ATT ATA TCA TCC TTG AGG ACA AGA CCG CAA CAG GTA AAA AGC GGG ATT GGA AGG Met Ile Ile Ser Ser Leu Arg Thr Arg Pro Gin Gln Vai Lys Ser Gly Ile Gly Arg 900
GGA Gly
AAA AGA GAC 6GA CGA ATC TTA TGG CTG AGC ACT ATG AAG CTT TAC AGA GTA AAA CTG Lys Arg Asp Giy Arg Ile Leu Trp Leu Ser Thr Met Lys Leu Tyr Arg Vai Lys Leu
GTA
950 TAC CTT ACT ATG CCA AAA AAG CTG AGA AAT TGT GCA GTT GTG COG AAT GTC TTT CGT Tyr Leu Thr Met Ala Lys Lys Leu Arg Asn Cys Ala Vai Val Arg Asn Vai Phe Arg
TTA
1000 AAC GAG ACC CGG AGA CGC AAA TTA AAG TTG TAT CAA GCT CAG TTT TOT AAA GTG AGG Asn Giu Thr Arg Arg Arg Lys Leu Lys Leu Tyr Gin Aua Gin Phe Cys Lys Vai Arg
105 TTA
0
TGC CCG ATG TGT GCG TGG CGT AGG TCT TTA AAA ATT GCT TAT CAT AAT AAA TTA ATC Cys Pro Met Cys Ala Trp Arg Arg Ser Leu Lys Ile Ala Tyr His Asn Lys Leu Ile
Vai
Leu Leu
1100
GTT Val
1150 GAG GAA GCG AAT CGG CAG TAC GGT TGT GGA TGG ATT TTT CTC ACA CTG ACG GTT CGG Glu Giu Aua Asn Arg Gln Tyr Giy Cys Giy Trp Ile Phe Leu Thr Leu Thr Vai Arg
AAT Asn
1 200 GTC GAG GGT GAC GGA TTA AAA CCC ATG ATT GCT GAC ATG ATG AAA GGA TGG AAC CGC Val Glu Gly Asp Gly Leu Lys Pro Met Ile Aua Asp Met Met Lys Gly Trp Asn Arg
CTT Leu
1 250 TTC GCA TAT AAA CGA GTT AAG GTA GCG ACT TTA GGT TAT TTC AGA GCT TTA GAG ATT Phe Gly Tyr Lys Arg Val Lys Val Aua Thr Leu Gly Tyr Phe Arg Au Leu Glu Ile
ACC Thr
1300 AAA AAT CAC GAA GAA GAT ACA TAT CAT CCG CAT TTT CAT GTG TTG TTG CCT GTG AAG Lys Asn His Giu Giu Asp Thr Tyr His Pro His Phe His Vii Leu Leu Pro Vii Lys
135 AAA Lys
0 1400 AGC TAT TTT ACT CAC AAT TAC ATT AAG CAG TCT GAG TGG ACG AGC TTA TGG AAA AGO GCG Ser Tyr Phe Thr His Asn Tyr Ile Lys Gin Ser Giu Trp Thr Ser Leu Trp Lys Arg Aua 1450 ATG AAA CTG GAC TAC ACG CCG ATT GTT GAT ATC CGA AGA GTC AAG GGA AGA OCT AAA Vai Lys Gly Arg Ala Lys
Met Lys Leu Asp Tyr Thr Pro Ile Val Asp Ile Arg Arg
1500 GAT GCC GAA CAG ATT GAG AGC OAT GTG COG GAA GCC ATG ATG GAO CAA AAA OCT GTT Asp Ala G1u Gn Ile Giu Ser Asp Val Arg G6u Ala Met Met Glu Gin Lys Ala Vai
OAA ATC TCT AAA TAT CCG Glu Ile Ser Lys Tyr Pro 1600
GAC AAT CTG AAC Asp Asn Leu Asn 0 TAC GOT C6C ATC Tyr Oly Gly Ile
OTT
1550
OAT
OAT OTT GTG CGC GGC Vil Val Arg Gly
ATT
Ile CTT
Leu
OAA
AAT AAG GTG ACA Thr Asn Lys
Olu
ACG GTG TTT TAT TTG OAT OAT GCG CTT TCT CGC CGC COO CTT ATT Thr Vai Phe Tyr Leou Asp Asp Ala Leu Ser Arg Arg Arg Lou Ile
Gly
Vai
AAA
Lys
TTG AAG GAA ATT Leu Lys
ACG
Asp Thr Asp
CAT AAA Lys
Glu Ile His
BAA CTA'AAC Olu Leou Asn
CTC
Leu
Vai
1700 GOT 6AT GCG GAG GAC GCC Sly Asp Ala O1u Asp Ghy
~~~~~~~~~~~~~~~1750OTT
* GAT CTC GTC AAG ATT GAO GAA GAA GAT GAC GAG GTG GCG AAC GAA GCA TTT GAA Asp Leu Vai Lys Ile Glu Olu Olu Asp Asp Glu Vai Ala Asn Gu Ala Phe Gu
1800
GCT TAC TOO CAT CCA GGC ATT AAA AAT TAC ATA ATC AGA TAA Via Tyr Trp His Pro Gly Ile Lys Asn Tyr Ile Ile Arg *** 1850
165
GOT
val
ATG
Met
TCAGATAAAAAGCAGGCGTTGTT 1900
CCTGCTTTTTTTATACTCTAATAGTCAAATCAAGAGTTAATTTTAGATGTAATTGTGAGAATTAGAGTGGCTGACCAGT 1950 ATTTGAAACTTCTTGGGCTACTTTCTTAACTTTATATTAAAACTATGTATATATGTGTTGTTTTTTCTATTATTTTOAT 2000 BstEII 2044 ATTATTTACAAGTATTGAAATTTTGCTAGGAGGGAAAGTTTTTATggttacc-3'
FIG. 3. Nucleotide sequence of the 2.0-kb BstEIl DNA fragment. -10, -35, and SD indicate, respectively, plausible sequence for the -10 and -35 sequences and the ribosome binding site. The nucleotide sequences indicated by lowercase latters at the two termini originate from the BstEII site.
1840
APPL. ENVIRON. MICROBIOL.
NOTES
pUH1, we used the trimethoprim-resistant (Tmpr) dihydrofolate reductase gene of B. subtilis 168. A schematic presentation of the constructed plasmids is given in Fig. 1. The dihydrofolate reductase-coding gene was derived from a Tmpr strain, TTK24, of B. subtilis 168 (20) and has been cloned in plasmid pBR322 of Escherichia coli (2). DNAs from pTL12, carrying the Tmpr dihydrofolate reductase gene, which was constructed by Tanaka and Kawano (18), and pBR322 were both treated with EcoRI and HindIII, mixed, and ligated with T4 ligase; then pATE1 was constructed. Natto plasmid pUH1 was digested with BstEII, and then the ends were filled in with Klenow fragment to generate a blunt end. The DNA fragments were mixed and ligated at the AatI site of pATE1 with T4 ligase and then added to B. subtilis MI112 (19) protoplasts. Several Tmpr colonies were obtained on AA agar plates (18) containing trimethoprim (1 ,ug/ml), and one of them was used for further study. Plasmid pBB2 carried in such a Tmpr colony had a molecular mass of 8.4 kb (Fig. 1). The physical map of pBB2, using various restriction enzymes, is shown in Fig. 1. To define the boundaries of a functional unit of pUH1 replication, a 2.0-kb BstEII fragment of pUH1 was digested with selected restriction endonucleases to obtain a set of overlapping DNA fragments. Digests modified with Klenow fragment were ligated with pATE1, followed by introduction into E. coli by transformation and successive selection for ampicillin resistance. The plasmid DNA preparations containing each generated fragment were tested for replication activity in B. subtilis. The results are summarized in Fig. 2. Plasmid pBB2, which corresponds to fragment 1 in Fig. 2, and a derivative containing fragment 7 specified Tmpr in B. subtilis, but attempts to transform B. subtilis with recombinant plasmid preparations containing the small fragments (fragments 2 to 6 in Fig. 2) and inserted fragments (fragments 8 and 9) were unsuccessful. The nucleotide sequence of the 2.0-kb BstEII fragment was determined by the method of Sangar et al. (14). Though the strategy is not shown, the nucleotide sequence was determined for both strands by using numerous restriction fragments to give enough overlapping regions. By examination of possible open reading frames, we found only one large frame (Fig. 2), designated rep, which consists of 999 bp and encodes a protein molecule with 333 amino acids and an Mr of 39,074. The 5' upstream region of rep contains a 5'-AAGGAG-3' sequence (indicated as SD in Fig. 3) complementary to the 3' end of 16S rRNA (3'-OH-UCUUUC CUCCAGUAG-5') of B. subtilis (11) at nucleotides 777 to 782 for translation initiation. There is a 5'-TATTAT-3' sequence (-10 in Fig. 3) resembling a Pribnow box (12) at nucleotides 727 to 732, and at a site 24 bp upstream of this -10 sequence, there is a 5'-TTGACA-3' sequence resembling the -35 consensus sequence (12) of the B. subtilis gene. The observed distance (18 bp) between the -10 and -35 sequences accords well with that observed generally in B. subtilis genes (17 to 18 bp) (12). Plasmid pBB2 was digested with BglII and then treated for 20 min with exonuclease Bal31 under conditions in which about 50 bp was removed per min from each end of the DNA molecule. After ligation by T4 ligase, the DNA was introduced into B. subtilis by protoplast transformation. Tmpr transformants, which contained fragment 11 in Fig. 2, were obtained at high efficiencies with Bal31-generated deletions of 581 bp (as determined by nucleotide sequencing), while no transformants were obtained with a similar 653-bp deletion (fragment 10 in Fig. 2), suggesting that the 5' upstream portion is dispensable for replication. The interaction of the
10
rep
repg
RepB Protein A
repg
RepB Protein A rep
rep,r RepB
30
40
50
60
100
110
120
--------
70
rep
20
------MIISSLRTRPQQVKSGIGRGKRDGRILWLSTMKLYRVKLVYLTMAKKLRNCAVV MYSSENDYRILEDKTATGKKRDWRGKKRRANLMAEHYEALEKRIGAPYYGKKAERLSECA ------------------------------------------MCYNMEKYTEKKQRNQVFQKFIKRHIGENQMDLVEDCN 80
90
RNVFRLNETRRRKLKLYQAQFCKVRLCPMCAWRRSLKIAYHNKLIVEEANRQYGCGWIFL EHLSFKRDPETGRLKLYQAHFCKVRLCPMCAWRRSLKIAYHNKLI IEEANRQYGCGWIFL -----------------------------------MKHGIQSQKVVAEVIKQKPTVRWLFL TFLSFVADKTLEKQKLYKANSCKNRFCPVCAWRKARKDALGLSLMMQYIKQQEKKEFIFL 130
140
150
160
170
180
TLTVRNV-EGDGLKPMIADMMKGWNRLFGYKRVKVATLGYFRALEITKNHEEDTYHPHFHV TLTVRNV-KGERLKPQISEMMEGFRKLFQYKKVKTSVLGFFRALEITKNHEEDTYHPHFHV TLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLVGFMRATEVTINNKDNSYNQHMHV
Protein A
TLTTPNVMSDE-LENEI KRYNNSFRKLIKRKKVGSVI KGYVRKLEITYNKKDNSYNQHMHV
rep
LLPVKKSYF--THNYIKQSEWTSLWKRAMKLDYTPIVDIRRVKGRAKIDAEQIESDVREAMM
RepB Protein A
LVCVEPTYFKNTENYVNGKQWIQFWKKAMKLDYDPNV-----------KVQMIRPKNKYKSD LIAVNKSYFTDKRYYISQQEWLDLW-----RDVTGISEITQVQVQKIRQNNNKELYEMAKYS
190
rep,,
200
210
220
230
240
LLPVKRNYF--GKNYI KQAEWTSLWKRAMKLDYTP IVDI RRVKGRVKIDAEQI ESDVREAMM 250
260
270
280
290
300
rep
rep,,
EQKAVLEISKYPVKDTDVVRGNKVTEDNLNTVFYLDDALSRRRLIGYGGILKEIHKELNL EQKAVLEISKYPVKTDDVVRGSKVTDDNLNTVFYLDDALSARRLIGYGGILKEIHKELNL
RepB Protein A
IQSAIDETAKYPVKDTD--FMTDDEEKNLKRLSDLEEGLHRKRLISYDGLLKEIHKKLNL --------------------------------------------GKDSDYLINKSKSL-
310 320 330 rep 6DAEDGDLVKIEEEDDEVANEAFEVMAYWHPGIKNYIIRrepe GDAEGGOLVKIEEEDDEVANGAFEVMAYWHPGIKNYILKRepB ODTEEGDL-IHTDDDEKADEDGFSIIAMWNWERKNYFIKE Protein A ----------------------------------------
FIG. 4. Comparison of the amino acid sequences of rep of pUH1, rep14 of pFTB14, RepB of pUB110, and protein A of pC194. Amino acid identities with rep of pUH1 are indicated (*). Amino
acid numbers follow the sequence of the rep protein from the amino-terminal methionine to the carboxyl terminus. Gaps have been inserted to gain maximum matching. The one-letter amino acid code has been used.
initiator protein with its binding site on the DNA is a key step in the replication initiation process (4, 21). Therefore, the structure of the 2.0-kb ori sequence that is necessary and sufficient for the replication of the Natto plasmid pUH1 molecule contains a 999-bp open reading frame, rep, a promotor region for rep expression, and a putative replication origin for the plasmid, which is located upstream of the promoter between positions 581 and 653. The amino acid sequence of the rep protein coding region of pUH1 was compared with a number of protein sequences registered in GenBank by using the homology search system of GENAS (10). Homologies between the predicted amino acid sequence of the rep protein of pUH1 and those of the rep protein of pFTB14, RepB of pUB110, and protein A of pC194 are illustrated in Fig. 4. A high similarity (71.8%) was found between the rep protein of pUH1 and repl4 of pFTB14 in B. amyloliquefaciens. The structure of the replication origin sequence of pFTB14 contains an open reading frame (rep), stretching for 1,017 bp, a promoter region for rep expression, and a possible replication origin for rep expression, which is located upstream of the promoter. The rep product is trans active and essential for plasmid replication (13). Compared with rep of pUH1, a low similarity (39.7%) was found for RepB of pUB110 (1) and for protein A (29.2%) of pC194 (8) in S. aureus. Khan et al. (9) determined the start site of pT181 DNA synthesis within a 127-bp segment and showed that a 168-bp segment containing the replication start site is enough to initiate unidirectional replication. Furthermore, like RepC protein of pT181 (9), the protein of E. coli plasmid R6K (4, 17, 18) does not have any significant homology in its amino acid sequences with those of the rep proteins of pUH1 and pFTB14 deduced from the open reading frame (data not shown). The Rep protein of Natto plasmid pUH1 shows high homology with replication proteins encoded by pFTB14, pC194, and pUB110, which originate from various gram-positive bacteria such as B. amyloliquefaciens and S. aureus. Taxonomic studies (based
VOL. 57, 1991
on the 16S rRNA similarities) revealed that Bacillus and S. aureus strains are more related than Streptococcus and Lactobacillus strains (16). The plasmid homologies suggest an exchange of plasmid replicons by recent horizontal transfer through the different genera, including "natto" Bacillus species. We are indebted to T. Tanaka for the generous supply of plasmid pTL12 and also to S. Kuhara for the computer search of protein sequences.
REFERENCES 1. Ano, T., T. Imanaka, and S. Aiba. 1986. The copy number of Bacillus plasmids pRBH1 is negatively controlled by RepB protein. Mol. Gen. Genet. 202:416-420. 2. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 3. Dagert, M., I. Jones, A. Goze, S. Romac, B. Niaudet, and S. D. Ehrlich. 1984. Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage. EMBO J. 3:81-86. 4. Germino, J., and D. Bastia. 1983. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell 34:125-134. 5. Hara, T., A. Aumayr, Y. Fujio, and S. Ueda. 1982. Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange. Appl. Environ. Microbiol. 44: 1456-1458. 6. Hara, T., A. Ishizaki, and S. Ueda. 1986. Formation of heteroduplex molecules between plasmids pUH1 and pLS11 in polyglutamate-producing Bacillus strains. Agric. Biol. Chem. 50: 2391-2394. 7. Horinouchi, S., and B. Weisblum. 1982. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150:815-825. 8. Iordanescu, S., and M. Surdeanu. 1983. Isolation and complementation of temperature-sensitive replication mutants of Staphylococcus aureus plasmid pC194. Mol. Gen. Genet. 191: 201-206. 9. Khan, S. H., S. M. Carlton, and R. P. Novick. 1982. Functional origin of replication of pT181 plasmid DNA is contained within
NOTES a
1841
168-base-pair segment. Proc. Natl. Acad. Sci. USA 79:4580-
4584. 10. Kuhara, S., F. Matsuo, S. Futamura, A. Fujita, T. Shinohara, T. Takagi, and Y. Sakaki. 1984. GENAS: a database system for nucleic acid sequence analysis. Nucleic Acids Res. 12:89-99. 11. McLaughlin, J. R., C. L. Murray, and J. C. Rabinowitz. 1981. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus P-lactamase gene. J. Biol. Chem. 256:11283-11291. 12. Moran, C. P., Jr, N. Lang, S. F. J. LeGrice, G. Lee, M. Stephans, A. L. Sonenshein, J. Pero, and R. Losick. 1982. Nucleotide sequence that signal the initiation of transcription and translation in Bacillus subtilis. Mol. Gen. Genet. 186:339346. 13. Murai, M., H. Miyashita, H. Araki, T. Seki, and Y. Oshima. 1987. Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14. Mol. Gen. Genet. 210:92100. 14. Sangar, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5476. 15. Scott, J. R. 1984. Regulation of plasmid replication. Microbiol. Rev. 48:1-23. 16. Stackebrandt, E., and M. Teuber. 1988. Molecular taxonomy and phylogenetic position of lactic acid bacteria. Biochimie 70:317-324. 17. Stalker, D. M., R. Kolter, and D. R. Helinski. 1982. Plasmid R6K DNA replication. I. Complete nucleotide sequence of an autonomously replicating segment. J. Mol. Biol. 161:33-43. 18. Tanaka, T., and N. Kawano. 1980. Cloning vehicles for the homologous Bacillus subtilis host-vector system. Gene 10:131136. 19. Tanaka, T., and K. Sakaguchi. 1978. Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168. Mol. Gen. Genet. 165:269-276. 20. Wainscott, V. J., and J. F. Kane. 1976. Dihydrofolate reductase in Bacillus subtilis, p. 208-213. In D. Schlessinger (ed.), Microbiology-1976. American Society for Microbiology, Washington, D.C. 21. Yamaguchi, K., and M. Yamaguchi. 1984. The replication origin of pSC101: the nucleotide sequence and replication functions of the ori region. Gene 29:211-219.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1838-1841
0099-2240/91/061838-04$02.00/0 Copyright C) 1991, American Society for Microbiology
Molecular Structure of the Replication Origin of a Bacillus subtilis (natto) Plasmid, pUHI TOSHIO HARA,* SINICHIRO NAGATOMO, SEIYA OGATA, AND SEINOSUKE UEDAt
Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan Received 8 November 1990/Accepted 7 March 1991
The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for y-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promotor region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with repl4 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.
fermented soybean food, natto. Recently, electron microscope studies of the hybridization products of pLS11, which is detected in B. subtilis IFO 3022 and is 8.6 kb in size, with pUH1 revealed a heteroduplex molecule containing 1.11and 2.14-kb double-stranded DNA termini (6). We thought that these double-strand regions might play an important role in polyglutamate production and DNA replication. This communication reports that the 2.0-kb BstEII DNA fragment corresponds to the replication origin of Natto plasmid pUH1 and contains a 999-bp open reading frame, a promoter for the open reading frame, and a possible replication origin upstream of the promoter. Significant homologies were observed between the amino acid sequence predicted from the 999-bp open reading frame and those of the similar putative proteins encoded by the B. amyloliquefaciens plasmid pFTB14 (13) and Staphylococcus aureus plasmid pC194 (3, 7). To facilitate identification of the replication region of
In a previous paper (5), we reported that a 5.7-kb plasmid designated pUH1, which encodes the y-glutamyltranspeptidase gene responsible for y-polyglutamate production, was detected in a Bacillus subtilis (natto) strain isolated from a
I 1000
Fragment 500
1
Phenotype 1750
1
2 3 4 5 6 7 8 8 9 10 11
FIG. 1. Derivation of plasmids used in the present study. The chain of solid circles in the diagram indicates the DNA segment containing the Tmpr gene of B. subtilis TTK24. Heavy and thin lines represent the regions of pBR322 and pTL12, respectively. Double
2000 bp +
+1
r
~~~~~v v
B B
4.
FIG. 2. Structure and replication activity of the modified fragments of the 2.0-kb ori fragment. The open arrow in the restriction map indicates an open reading frame found in the sequence data (see Fig. 3). + and - indicate, respectively, ability and inability to replicate in the B. subtilis host. Modifications were made by deletion with restriction enzymes or insertion of a few base pairs (closed triangle), using the Klenow fragment, after digestion with the indicated restriction enzyme or by Bal31 digestion (B) from the BglII
lines represent the DNA fragment of pUH1. A, AatI; Bg, BglII; Bs, BstELL; E, EcoRI; H, HindIII; P, PstI.
* Corresponding author. t Present address: Department of Applied Microbiology and Technology, Kumamoto Institute of Technology, Ikeda, Kumamoto 860, Japan.
site of pBB2. 1838
VOL. 57, 1991
NOTES
1839
BstE II
5' -ggtaaccGGACCGTAGGGA 50
1TA GGATTAAGGAAGTTGACTCGCTCAGCGCCACCCGAACCCTTTCCGCACTCt.AACAAACCCGTTTGTTTGACGCCAACCG 150
°°
GGCAGGGAGCCCCCCGAAGAAGCGGGGGTTGGGGGGATTGAATGCTGGCATCCAACGGCCGTCCGTTGGTGGGTTTGGG 250
200
CAAAGCCAAGAACTGTTGCAAGGCTCGTTGAGAATAAAGAATGCTTTTCAGGATGCTTAGAATCGTTTCTGAGAGCTTC 300
AAATAAAAAAGATGACCTTTTATAGGGGGAAGCTCTTAAAATTGAATGTAGGGGCATTTAAACACGTTTAAAAATAAAA 350
400
AAAGCAGACTCTTTAGAGTCCGCCTTGTTATTTTTAACCCAGTGCTCCATTTTCGGCTGTTTGGAAATCTTTTGAGATG CCGAACCATCCATTTTCTTTTGTTCCATGAAAAAAGTGCTTTTGGATGCTTAAAAAGGCTTTTTCGTATAAAAAAAGCC 500
550
GATTTTTGAAAAAAAAATCTCCCTGCGGGGGAAGAATGGTTTTGATCTTTGGGTTTTAGGTTTTAAAAAAACCGGGCTG 650
600
TTTTCAGCCGGCTTTTTTTCGATTTTGGCGGAGCCGAAATCGGGTCTTTTCTTATCTTGATACTATATAGAAACATCTC 2
12
36
AAGGCGAAAAAATAGCCCCCATCCCTTATTTGTCAAGGGTTTGACGGCTTTGTTAGAAACTCCTTCCGCffl (SD) so '800 M3AAAGTGCCCACTAAAATAATAGAATGCTAGATTACTAG CTCA q~ TTTTTTGT6TCATGTATTCATCTGAAA 850 ATG ATT ATA TCA TCC TTG AGG ACA AGA CCG CAA CAG GTA AAA AGC GGG ATT GGA AGG Met Ile Ile Ser Ser Leu Arg Thr Arg Pro Gin Gln Vai Lys Ser Gly Ile Gly Arg 900
GGA Gly
AAA AGA GAC 6GA CGA ATC TTA TGG CTG AGC ACT ATG AAG CTT TAC AGA GTA AAA CTG Lys Arg Asp Giy Arg Ile Leu Trp Leu Ser Thr Met Lys Leu Tyr Arg Vai Lys Leu
GTA
950 TAC CTT ACT ATG CCA AAA AAG CTG AGA AAT TGT GCA GTT GTG COG AAT GTC TTT CGT Tyr Leu Thr Met Ala Lys Lys Leu Arg Asn Cys Ala Vai Val Arg Asn Vai Phe Arg
TTA
1000 AAC GAG ACC CGG AGA CGC AAA TTA AAG TTG TAT CAA GCT CAG TTT TOT AAA GTG AGG Asn Giu Thr Arg Arg Arg Lys Leu Lys Leu Tyr Gin Aua Gin Phe Cys Lys Vai Arg
105 TTA
0
TGC CCG ATG TGT GCG TGG CGT AGG TCT TTA AAA ATT GCT TAT CAT AAT AAA TTA ATC Cys Pro Met Cys Ala Trp Arg Arg Ser Leu Lys Ile Ala Tyr His Asn Lys Leu Ile
Vai
Leu Leu
1100
GTT Val
1150 GAG GAA GCG AAT CGG CAG TAC GGT TGT GGA TGG ATT TTT CTC ACA CTG ACG GTT CGG Glu Giu Aua Asn Arg Gln Tyr Giy Cys Giy Trp Ile Phe Leu Thr Leu Thr Vai Arg
AAT Asn
1 200 GTC GAG GGT GAC GGA TTA AAA CCC ATG ATT GCT GAC ATG ATG AAA GGA TGG AAC CGC Val Glu Gly Asp Gly Leu Lys Pro Met Ile Aua Asp Met Met Lys Gly Trp Asn Arg
CTT Leu
1 250 TTC GCA TAT AAA CGA GTT AAG GTA GCG ACT TTA GGT TAT TTC AGA GCT TTA GAG ATT Phe Gly Tyr Lys Arg Val Lys Val Aua Thr Leu Gly Tyr Phe Arg Au Leu Glu Ile
ACC Thr
1300 AAA AAT CAC GAA GAA GAT ACA TAT CAT CCG CAT TTT CAT GTG TTG TTG CCT GTG AAG Lys Asn His Giu Giu Asp Thr Tyr His Pro His Phe His Vii Leu Leu Pro Vii Lys
135 AAA Lys
0 1400 AGC TAT TTT ACT CAC AAT TAC ATT AAG CAG TCT GAG TGG ACG AGC TTA TGG AAA AGO GCG Ser Tyr Phe Thr His Asn Tyr Ile Lys Gin Ser Giu Trp Thr Ser Leu Trp Lys Arg Aua 1450 ATG AAA CTG GAC TAC ACG CCG ATT GTT GAT ATC CGA AGA GTC AAG GGA AGA OCT AAA Vai Lys Gly Arg Ala Lys
Met Lys Leu Asp Tyr Thr Pro Ile Val Asp Ile Arg Arg
1500 GAT GCC GAA CAG ATT GAG AGC OAT GTG COG GAA GCC ATG ATG GAO CAA AAA OCT GTT Asp Ala G1u Gn Ile Giu Ser Asp Val Arg G6u Ala Met Met Glu Gin Lys Ala Vai
OAA ATC TCT AAA TAT CCG Glu Ile Ser Lys Tyr Pro 1600
GAC AAT CTG AAC Asp Asn Leu Asn 0 TAC GOT C6C ATC Tyr Oly Gly Ile
OTT
1550
OAT
OAT OTT GTG CGC GGC Vil Val Arg Gly
ATT
Ile CTT
Leu
OAA
AAT AAG GTG ACA Thr Asn Lys
Olu
ACG GTG TTT TAT TTG OAT OAT GCG CTT TCT CGC CGC COO CTT ATT Thr Vai Phe Tyr Leou Asp Asp Ala Leu Ser Arg Arg Arg Lou Ile
Gly
Vai
AAA
Lys
TTG AAG GAA ATT Leu Lys
ACG
Asp Thr Asp
CAT AAA Lys
Glu Ile His
BAA CTA'AAC Olu Leou Asn
CTC
Leu
Vai
1700 GOT 6AT GCG GAG GAC GCC Sly Asp Ala O1u Asp Ghy
~~~~~~~~~~~~~~~1750OTT
* GAT CTC GTC AAG ATT GAO GAA GAA GAT GAC GAG GTG GCG AAC GAA GCA TTT GAA Asp Leu Vai Lys Ile Glu Olu Olu Asp Asp Glu Vai Ala Asn Gu Ala Phe Gu
1800
GCT TAC TOO CAT CCA GGC ATT AAA AAT TAC ATA ATC AGA TAA Via Tyr Trp His Pro Gly Ile Lys Asn Tyr Ile Ile Arg *** 1850
165
GOT
val
ATG
Met
TCAGATAAAAAGCAGGCGTTGTT 1900
CCTGCTTTTTTTATACTCTAATAGTCAAATCAAGAGTTAATTTTAGATGTAATTGTGAGAATTAGAGTGGCTGACCAGT 1950 ATTTGAAACTTCTTGGGCTACTTTCTTAACTTTATATTAAAACTATGTATATATGTGTTGTTTTTTCTATTATTTTOAT 2000 BstEII 2044 ATTATTTACAAGTATTGAAATTTTGCTAGGAGGGAAAGTTTTTATggttacc-3'
FIG. 3. Nucleotide sequence of the 2.0-kb BstEIl DNA fragment. -10, -35, and SD indicate, respectively, plausible sequence for the -10 and -35 sequences and the ribosome binding site. The nucleotide sequences indicated by lowercase latters at the two termini originate from the BstEII site.
1840
APPL. ENVIRON. MICROBIOL.
NOTES
pUH1, we used the trimethoprim-resistant (Tmpr) dihydrofolate reductase gene of B. subtilis 168. A schematic presentation of the constructed plasmids is given in Fig. 1. The dihydrofolate reductase-coding gene was derived from a Tmpr strain, TTK24, of B. subtilis 168 (20) and has been cloned in plasmid pBR322 of Escherichia coli (2). DNAs from pTL12, carrying the Tmpr dihydrofolate reductase gene, which was constructed by Tanaka and Kawano (18), and pBR322 were both treated with EcoRI and HindIII, mixed, and ligated with T4 ligase; then pATE1 was constructed. Natto plasmid pUH1 was digested with BstEII, and then the ends were filled in with Klenow fragment to generate a blunt end. The DNA fragments were mixed and ligated at the AatI site of pATE1 with T4 ligase and then added to B. subtilis MI112 (19) protoplasts. Several Tmpr colonies were obtained on AA agar plates (18) containing trimethoprim (1 ,ug/ml), and one of them was used for further study. Plasmid pBB2 carried in such a Tmpr colony had a molecular mass of 8.4 kb (Fig. 1). The physical map of pBB2, using various restriction enzymes, is shown in Fig. 1. To define the boundaries of a functional unit of pUH1 replication, a 2.0-kb BstEII fragment of pUH1 was digested with selected restriction endonucleases to obtain a set of overlapping DNA fragments. Digests modified with Klenow fragment were ligated with pATE1, followed by introduction into E. coli by transformation and successive selection for ampicillin resistance. The plasmid DNA preparations containing each generated fragment were tested for replication activity in B. subtilis. The results are summarized in Fig. 2. Plasmid pBB2, which corresponds to fragment 1 in Fig. 2, and a derivative containing fragment 7 specified Tmpr in B. subtilis, but attempts to transform B. subtilis with recombinant plasmid preparations containing the small fragments (fragments 2 to 6 in Fig. 2) and inserted fragments (fragments 8 and 9) were unsuccessful. The nucleotide sequence of the 2.0-kb BstEII fragment was determined by the method of Sangar et al. (14). Though the strategy is not shown, the nucleotide sequence was determined for both strands by using numerous restriction fragments to give enough overlapping regions. By examination of possible open reading frames, we found only one large frame (Fig. 2), designated rep, which consists of 999 bp and encodes a protein molecule with 333 amino acids and an Mr of 39,074. The 5' upstream region of rep contains a 5'-AAGGAG-3' sequence (indicated as SD in Fig. 3) complementary to the 3' end of 16S rRNA (3'-OH-UCUUUC CUCCAGUAG-5') of B. subtilis (11) at nucleotides 777 to 782 for translation initiation. There is a 5'-TATTAT-3' sequence (-10 in Fig. 3) resembling a Pribnow box (12) at nucleotides 727 to 732, and at a site 24 bp upstream of this -10 sequence, there is a 5'-TTGACA-3' sequence resembling the -35 consensus sequence (12) of the B. subtilis gene. The observed distance (18 bp) between the -10 and -35 sequences accords well with that observed generally in B. subtilis genes (17 to 18 bp) (12). Plasmid pBB2 was digested with BglII and then treated for 20 min with exonuclease Bal31 under conditions in which about 50 bp was removed per min from each end of the DNA molecule. After ligation by T4 ligase, the DNA was introduced into B. subtilis by protoplast transformation. Tmpr transformants, which contained fragment 11 in Fig. 2, were obtained at high efficiencies with Bal31-generated deletions of 581 bp (as determined by nucleotide sequencing), while no transformants were obtained with a similar 653-bp deletion (fragment 10 in Fig. 2), suggesting that the 5' upstream portion is dispensable for replication. The interaction of the
10
rep
repg
RepB Protein A
repg
RepB Protein A rep
rep,r RepB
30
40
50
60
100
110
120
--------
70
rep
20
------MIISSLRTRPQQVKSGIGRGKRDGRILWLSTMKLYRVKLVYLTMAKKLRNCAVV MYSSENDYRILEDKTATGKKRDWRGKKRRANLMAEHYEALEKRIGAPYYGKKAERLSECA ------------------------------------------MCYNMEKYTEKKQRNQVFQKFIKRHIGENQMDLVEDCN 80
90
RNVFRLNETRRRKLKLYQAQFCKVRLCPMCAWRRSLKIAYHNKLIVEEANRQYGCGWIFL EHLSFKRDPETGRLKLYQAHFCKVRLCPMCAWRRSLKIAYHNKLI IEEANRQYGCGWIFL -----------------------------------MKHGIQSQKVVAEVIKQKPTVRWLFL TFLSFVADKTLEKQKLYKANSCKNRFCPVCAWRKARKDALGLSLMMQYIKQQEKKEFIFL 130
140
150
160
170
180
TLTVRNV-EGDGLKPMIADMMKGWNRLFGYKRVKVATLGYFRALEITKNHEEDTYHPHFHV TLTVRNV-KGERLKPQISEMMEGFRKLFQYKKVKTSVLGFFRALEITKNHEEDTYHPHFHV TLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLVGFMRATEVTINNKDNSYNQHMHV
Protein A
TLTTPNVMSDE-LENEI KRYNNSFRKLIKRKKVGSVI KGYVRKLEITYNKKDNSYNQHMHV
rep
LLPVKKSYF--THNYIKQSEWTSLWKRAMKLDYTPIVDIRRVKGRAKIDAEQIESDVREAMM
RepB Protein A
LVCVEPTYFKNTENYVNGKQWIQFWKKAMKLDYDPNV-----------KVQMIRPKNKYKSD LIAVNKSYFTDKRYYISQQEWLDLW-----RDVTGISEITQVQVQKIRQNNNKELYEMAKYS
190
rep,,
200
210
220
230
240
LLPVKRNYF--GKNYI KQAEWTSLWKRAMKLDYTP IVDI RRVKGRVKIDAEQI ESDVREAMM 250
260
270
280
290
300
rep
rep,,
EQKAVLEISKYPVKDTDVVRGNKVTEDNLNTVFYLDDALSRRRLIGYGGILKEIHKELNL EQKAVLEISKYPVKTDDVVRGSKVTDDNLNTVFYLDDALSARRLIGYGGILKEIHKELNL
RepB Protein A
IQSAIDETAKYPVKDTD--FMTDDEEKNLKRLSDLEEGLHRKRLISYDGLLKEIHKKLNL --------------------------------------------GKDSDYLINKSKSL-
310 320 330 rep 6DAEDGDLVKIEEEDDEVANEAFEVMAYWHPGIKNYIIRrepe GDAEGGOLVKIEEEDDEVANGAFEVMAYWHPGIKNYILKRepB ODTEEGDL-IHTDDDEKADEDGFSIIAMWNWERKNYFIKE Protein A ----------------------------------------
FIG. 4. Comparison of the amino acid sequences of rep of pUH1, rep14 of pFTB14, RepB of pUB110, and protein A of pC194. Amino acid identities with rep of pUH1 are indicated (*). Amino
acid numbers follow the sequence of the rep protein from the amino-terminal methionine to the carboxyl terminus. Gaps have been inserted to gain maximum matching. The one-letter amino acid code has been used.
initiator protein with its binding site on the DNA is a key step in the replication initiation process (4, 21). Therefore, the structure of the 2.0-kb ori sequence that is necessary and sufficient for the replication of the Natto plasmid pUH1 molecule contains a 999-bp open reading frame, rep, a promotor region for rep expression, and a putative replication origin for the plasmid, which is located upstream of the promoter between positions 581 and 653. The amino acid sequence of the rep protein coding region of pUH1 was compared with a number of protein sequences registered in GenBank by using the homology search system of GENAS (10). Homologies between the predicted amino acid sequence of the rep protein of pUH1 and those of the rep protein of pFTB14, RepB of pUB110, and protein A of pC194 are illustrated in Fig. 4. A high similarity (71.8%) was found between the rep protein of pUH1 and repl4 of pFTB14 in B. amyloliquefaciens. The structure of the replication origin sequence of pFTB14 contains an open reading frame (rep), stretching for 1,017 bp, a promoter region for rep expression, and a possible replication origin for rep expression, which is located upstream of the promoter. The rep product is trans active and essential for plasmid replication (13). Compared with rep of pUH1, a low similarity (39.7%) was found for RepB of pUB110 (1) and for protein A (29.2%) of pC194 (8) in S. aureus. Khan et al. (9) determined the start site of pT181 DNA synthesis within a 127-bp segment and showed that a 168-bp segment containing the replication start site is enough to initiate unidirectional replication. Furthermore, like RepC protein of pT181 (9), the protein of E. coli plasmid R6K (4, 17, 18) does not have any significant homology in its amino acid sequences with those of the rep proteins of pUH1 and pFTB14 deduced from the open reading frame (data not shown). The Rep protein of Natto plasmid pUH1 shows high homology with replication proteins encoded by pFTB14, pC194, and pUB110, which originate from various gram-positive bacteria such as B. amyloliquefaciens and S. aureus. Taxonomic studies (based
VOL. 57, 1991
on the 16S rRNA similarities) revealed that Bacillus and S. aureus strains are more related than Streptococcus and Lactobacillus strains (16). The plasmid homologies suggest an exchange of plasmid replicons by recent horizontal transfer through the different genera, including "natto" Bacillus species. We are indebted to T. Tanaka for the generous supply of plasmid pTL12 and also to S. Kuhara for the computer search of protein sequences.
REFERENCES 1. Ano, T., T. Imanaka, and S. Aiba. 1986. The copy number of Bacillus plasmids pRBH1 is negatively controlled by RepB protein. Mol. Gen. Genet. 202:416-420. 2. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 3. Dagert, M., I. Jones, A. Goze, S. Romac, B. Niaudet, and S. D. Ehrlich. 1984. Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage. EMBO J. 3:81-86. 4. Germino, J., and D. Bastia. 1983. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell 34:125-134. 5. Hara, T., A. Aumayr, Y. Fujio, and S. Ueda. 1982. Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange. Appl. Environ. Microbiol. 44: 1456-1458. 6. Hara, T., A. Ishizaki, and S. Ueda. 1986. Formation of heteroduplex molecules between plasmids pUH1 and pLS11 in polyglutamate-producing Bacillus strains. Agric. Biol. Chem. 50: 2391-2394. 7. Horinouchi, S., and B. Weisblum. 1982. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150:815-825. 8. Iordanescu, S., and M. Surdeanu. 1983. Isolation and complementation of temperature-sensitive replication mutants of Staphylococcus aureus plasmid pC194. Mol. Gen. Genet. 191: 201-206. 9. Khan, S. H., S. M. Carlton, and R. P. Novick. 1982. Functional origin of replication of pT181 plasmid DNA is contained within
NOTES a
1841
168-base-pair segment. Proc. Natl. Acad. Sci. USA 79:4580-
4584. 10. Kuhara, S., F. Matsuo, S. Futamura, A. Fujita, T. Shinohara, T. Takagi, and Y. Sakaki. 1984. GENAS: a database system for nucleic acid sequence analysis. Nucleic Acids Res. 12:89-99. 11. McLaughlin, J. R., C. L. Murray, and J. C. Rabinowitz. 1981. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus P-lactamase gene. J. Biol. Chem. 256:11283-11291. 12. Moran, C. P., Jr, N. Lang, S. F. J. LeGrice, G. Lee, M. Stephans, A. L. Sonenshein, J. Pero, and R. Losick. 1982. Nucleotide sequence that signal the initiation of transcription and translation in Bacillus subtilis. Mol. Gen. Genet. 186:339346. 13. Murai, M., H. Miyashita, H. Araki, T. Seki, and Y. Oshima. 1987. Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14. Mol. Gen. Genet. 210:92100. 14. Sangar, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5476. 15. Scott, J. R. 1984. Regulation of plasmid replication. Microbiol. Rev. 48:1-23. 16. Stackebrandt, E., and M. Teuber. 1988. Molecular taxonomy and phylogenetic position of lactic acid bacteria. Biochimie 70:317-324. 17. Stalker, D. M., R. Kolter, and D. R. Helinski. 1982. Plasmid R6K DNA replication. I. Complete nucleotide sequence of an autonomously replicating segment. J. Mol. Biol. 161:33-43. 18. Tanaka, T., and N. Kawano. 1980. Cloning vehicles for the homologous Bacillus subtilis host-vector system. Gene 10:131136. 19. Tanaka, T., and K. Sakaguchi. 1978. Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168. Mol. Gen. Genet. 165:269-276. 20. Wainscott, V. J., and J. F. Kane. 1976. Dihydrofolate reductase in Bacillus subtilis, p. 208-213. In D. Schlessinger (ed.), Microbiology-1976. American Society for Microbiology, Washington, D.C. 21. Yamaguchi, K., and M. Yamaguchi. 1984. The replication origin of pSC101: the nucleotide sequence and replication functions of the ori region. Gene 29:211-219.
